Exemestane (EXE) is a hormonal therapy used to treat estrogen receptor–positive breast cancer by inhibiting the final step of estrogen biosynthesis catalyzed by the enzyme aromatase. Cysteine conjugates of EXE and its active metabolite 17β-dihydro-EXE (DHE) are the major metabolites found in both the urine and plasma of patients taking EXE. The initial step in cysteine conjugate formation is glutathione conjugation catalyzed by the glutathione S -transferase (GST) family of enzymes. The goal of the present study was to identify cytosolic hepatic GSTs active in the GST-mediated metabolism of EXE and 17β-DHE. Twelve recombinant cytosolic hepatic GSTs were screened for their activity against EXE and 17β-DHE, and glutathionylated EXE and 17β-DHE conjugates were detected by ultra-performance liquid chromatography tandem mass spectrometry. GST α (GSTA) isoform 1, GST μ (GSTM) isoform 3 and isoform 1 were active against EXE, whereas only GSTA1 exhibited activity against 17β-DHE. GSTM1 exhibited the highest affinity against EXE with a Michaelis-Menten constant (K M ) value that was 3.8- and 7.1-fold lower than that observed for GSTA1 and GSTM3, respectively. Of the three GSTs, GSTM3 exhibited the highest intrinsic clearance against EXE (intrinsic clearance = 0.14 nl·min −1 ·mg −1 ). The K M values observed for human liver cytosol against EXE (46 μM) and 17β-DHE (77 μM) were similar to those observed for recombinant GSTA1 (53 and 30 μM, respectively). Western blot analysis revealed that GSTA1 and GSTM1 composed 4.3% and 0.57%, respectively, of total protein in human liver cytosol; GSTM3 was not detected. These data suggest that GSTA1 is the major hepatic cytosolic enzyme involved in the clearance of EXE and its major active metabolite, 17β-DHE. SIGNIFICANCE STATEMENT Most previous studies related to the metabolism of the aromatase inhibitor exemestane (EXE) have focused mainly on phase I metabolic pathways and the glucuronidation phase II metabolic pathway. However, recent studies have indicated that glutathionylation is the major metabolic pathway for EXE. The present study is the first to characterize hepatic glutathione S -transferase (GST) activity against EXE and 17β-dihydro-EXE and to identify GST α 1 and GST μ 1 as the major cytosolic GSTs involved in the hepatic metabolism of EXE.

Exemestane (EXE) is used to treat postmenopausal women diagnosed with estrogen receptor positive (ER+) breast cancer. A major mode of metabolism of EXE and its active metabolite, 17 β -dihydroexemestane, is via glutathionylation by glutathione-S-transferase (GST) enzymes. The goal of the present study was to investigate the effects of genetic variation in EXE-metabolizing GST enzymes on overall EXE metabolism. Ex vivo assays examining human liver cytosols from 75 subjects revealed the GSTA1 *B*B genotype was associated with significant decreases in S-(androsta-1,4-diene-3,17-dion-6 α -ylmethyl)-L-glutathione ( P = 0.034) and S-(androsta-1,4-diene-17 β -ol-3-on-6 α -ylmethyl)-L-gutathione ( P = 0.014) formation. In the plasma of 68 ER+ breast cancer patients treated with EXE, the GSTA1 *B*B genotype was associated with significant decreases in both EXE-cysteine (cys) (29%, P = 0.0056) and 17 β -DHE-cys (34%, P = 0.032) as compared with patients with the GSTA1*A*A genotype, with significant decreases in EXE-cys ( P trend = 0.0067) and 17 β -DHE-cys ( P trend = 0.028) observed in patients with increasing numbers of the GSTA1*B allele. A near-significant ( P trend = 0.060) trend was also observed for urinary EXE-cys levels from the same patients. In contrast, plasma and urinary 17 β -DHE-Gluc levels were significantly increased (36%, P = 0.00097 and 52%, P = 0.0089; respectively) in patients with the GSTA1 *B*B genotype. No significant correlations were observed between the GSTM1 null genotype and EXE metabolite levels. These data suggest that the GSTA1*B allele is associated with interindividual differences in EXE metabolism and may play a role in interindividual variability in overall response to EXE. SIGNIFICANCE STATEMENT The present study is the first comprehensive pharmacogenomic investigation examining the role of genetic variability in GST enzymes on exemestane metabolism. The GSTA1 *B*B genotype was found to contribute to interindividual differences in the metabolism of EXE both ex vivo and in clinical samples from patients taking EXE for the treatment of ER+ breast cancer. Since GSTA1 is a major hepatic phase II metabolizing enzyme in EXE metabolism, the GSTA1*B allele may be an important biomarker for treatment outcomes and toxicities.

Breast Cancer is the second leading cause of cancer death among women in the USA, with estrogen‐receptor positive (ER+) tumors accounting for 75% of breast cancers in postmenopausal women. Endocrine therapy targeting the ER pathway is a major treatment modality for ER+ patients. Exemestane (EXE) is a third generation aromatase inhibitor which blocks the aromatase enzyme in the final step of estrogen biosynthesis. Studies examining the long‐term use of EXE as a chemopreventive agent demonstrated a reduction in breast cancer incidence by more than 65%. EXE represents an improvement in breast cancer therapy, but inter‐individual variability exists in overall patient response and adverse events that may be attributed to variation in EXE metabolism. Preliminary studies have demonstrated that cysteine conjugates of EXE and its active metabolite 17β‐dihydro‐EXE (17β‐DHE) comprise 77% of total EXE metabolites in the urine of subjects taking EXE. The initial step in cysteine conjugate formation is glutathione (GSH) conjugation in a reaction catalyzed by the glutathione‐S‐transferase (GST) family of the enzymes involved in the metabolism of a variety of exogenous and endogenous substances. The goal of the present study was to identify hepatic GSTs active in the metabolism of EXE and 17β‐DHE. In a screening of the Human Protein Atlas, a total of 12 cytosolic and 3 microsomal GSTs were found to be hepatically expressed. Commercially available cytosolic GSTs were purchased, and their activity was verified with a common GST substrate, 1‐chloro‐2.4‐dinitrobenzene (CDNB). Additionally, GSTT1, GSTO1, GSTZ1, GSTA1, GSTM3, and GSTP1 were produced as recombinant histidine‐tagged proteins using an E‐coli expression system and found to be 97–99% pure by silver staining. Microsomal GSTs were cloned with a V5 epitope tag, overexpressed in the HEK293 cell line, and verified by Western Blot analysis using an anti‐V5 antibody. The 12 cytosolic and 3 microsomal GSTs were screened by incubating GSH with EXE or 17β‐DHE (0.125mM) using 0.5 μg of pure recombinant protein or 20 μg of microsomal protein. GS‐conjugates of EXE and DHE were detected using ultra‐performance liquid chromatography combined with tandem mass spectrometry (UPLC‐MS/MS). Results from the individual EXE‐GSH conjugation assays indicate that the cytosolic enzymes GSTA1, GSTM3, GSTP1 and GSTT1 are active against EXE and marginally active against 17β‐DHE, with GSTA1 exhibiting the highest relative activity. The three screened microsomal MGST1, MGST2 and MGST3 all exhibit activity against both EXE and 17β‐DHE, with MGST2 exhibiting the highest activity against 17β‐DHE. These data suggest that both cytosolic and microsomal GSTs are responsible for EXE‐GS and 17β‐DHE‐GS conjugate formation in vitro and that GSH conjugation by GSTs may be an important metabolic pathway contributing to excretion and elimination of EXE in people.Support or Funding InformationThis work is supported by grants from NIH, National Cancer Institute (grant R01‐ CA164366; to P. Lazarus) and the Health Sciences and Services Authority of Spokane, Washington (grant WSU002292 to College of Pharmacy and Pharmaceutical Sciences, Washington State University)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Breast Cancer is the second leading cause of cancer death among women in the USA, with estrogen‐receptor positive (ER+) tumors accounting for 75% of breast cancers in postmenopausal women. Endocrine therapy targeting the ER pathway is a major treatment modality for ER+ patients. Exemestane (EXE) is a third generation aromatase inhibitor which blocks the aromatase enzyme in the final step of estrogen biosynthesis. Studies examining the long‐term use of EXE as a chemopreventive agent demonstrated a reduction in breast cancer incidence by more than 65%. EXE represents an improvement in breast cancer therapy, but inter‐individual variability exists in overall patient response and adverse events that may be attributed to variation in EXE metabolism. Preliminary studies have demonstrated that cysteine conjugates of EXE and its active metabolite 17β‐dihydro‐EXE (17β‐DHE) comprise 77% of total EXE metabolites in the urine of subjects taking EXE. The initial step in cysteine conjugate formation is glutathione (GSH) conjugation in a reaction catalyzed by the glutathione‐S‐transferase (GST) family of the enzymes involved in the metabolism of a variety of exogenous and endogenous substances. The goal of the present study was to identify hepatic GSTs active in the metabolism of EXE and 17β‐DHE. In a screening of the Human Protein Atlas, a total of 12 cytosolic and 3 microsomal GSTs were found to be hepatically expressed. Commercially available cytosolic GSTs were purchased, and their activity was verified with a common GST substrate, 1‐chloro‐2.4‐dinitrobenzene (CDNB). Additionally, GSTT1, GSTO1, GSTZ1, GSTA1, GSTM3, and GSTP1 were produced as recombinant histidine‐tagged proteins using an E‐coli expression system and found to be 97–99% pure by silver staining. Microsomal GSTs were cloned with a V5 epitope tag, overexpressed in the HEK293 cell line, and verified by Western Blot analysis using an anti‐V5 antibody. The 12 cytosolic and 3 microsomal GSTs were screened by incubating GSH with EXE or 17β‐DHE (0.125mM) using 0.5 μg of pure recombinant protein or 20 μg of microsomal protein. GS‐conjugates of EXE and DHE were detected using ultra‐performance liquid chromatography combined with tandem mass spectrometry (UPLC‐MS/MS). Results from the individual EXE‐GSH conjugation assays indicate that the cytosolic enzymes GSTA1, GSTM3, GSTP1 and GSTT1 are active against EXE and marginally active against 17β‐DHE, with GSTA1 exhibiting the highest relative activity. The three screened microsomal MGST1, MGST2 and MGST3 all exhibit activity against both EXE and 17β‐DHE, with MGST2 exhibiting the highest activity against 17β‐DHE. These data suggest that both cytosolic and microsomal GSTs are responsible for EXE‐GS and 17β‐DHE‐GS conjugate formation in vitro and that GSH conjugation by GSTs may be an important metabolic pathway contributing to excretion and elimination of EXE in people. Support or Funding Information This work is supported by grants from NIH, National Cancer Institute (grant R01‐ CA164366; to P. Lazarus) and the Health Scien...